Fig. 7. Antibody uptake assay shows that phosphorylated kAE1 rapidly internalises from the plasma membrane in nonpolarised cells. MDCKI-kAE1, MDCKI-kAE1Y359A or MDCKI-Nt1-360AE1 cells were seeded onto coverslips and an antibody-uptake assay using FITC-Bric6 conducted under various conditions. (A-H) Pervanadate induces kAE1 internalisation. The majority of the kAE1 FITC-Bric6 is at the cell surface before internalisation (A,B). (C,D) FITC-Bric6 internalisation at 37°C for 30 minutes in the absence and presence of pervanadate (PV). (E-H) FITC-Bric6 is susceptible to acid wash in control cells (compare E and F) but pervanadate-treated cells are resistant to this process (compare G and H), consistent with the majority of the kAE1 having been internalised. (I-K) Hypertonicity inhibits kAE1 internalisation. (I) There is a lack of FITC-Bric6 internalisation following 1 hour exposure to 514 mM NaHCO₃. (J,K) In the presence of 514 mM NaCl or 1 M sucrose, pervanadate-induced FITC-Bric6 internalisation is inhibited. (L-T) kAE1 mutants that lack Y359 have a rapid internalisation of FITC-Bric6 and this is inhibited by Src kinase inhibitor PP2. (L) FITC-Bric6 binding to kAE1Y359A cells before internalisation is allowed to commence. (M,N) kAE1Y359A uptake in the absence or presence of pervanadate. This demonstrates that Y359A rapidly internalises and there is no obvious difference between antibody uptake in the presence or absence of pervanadate. (O-T) Comparison of kAE1Y359A (O and P), Nt1-360AE1 (Q and R) or kAE1 (S and T) treated with either PP3 or PP2 as indicated. Scale bar: 30 µm.